Phospholipid transfer proteins capable of exchanging phospholipids between membrane bilayers in vitro have been extensively characterized, but the role of these proteins in vivo has been more difficult to establish. See, for example, Cleves, A., et al., Cell 64:789 (1991), the disclosure of which is incorporated herein by reference. (Hereafter all additional citations of a particular publication shall be to the author(s) and year only.) A breakthrough occurred with the discovery that the SEC14 gene of Saccharomyces cerevisiae encodes the phosphatidylinositol (PI)/phosphatidylcholine (PC) transporter/transfer protein which is essential to the secretary pathway and to viability as reported by Bankaitis, V., et al., Nature 347:561 (1990), the disclosure of which is incorporated herein by reference. Deletion of the yeast SEC14 gene in an otherwise wild type cell is lethal and inactivation of the SEC14 gene product in a temperature-sensitive (sec14.sup.ts) mutant leads to arrest of the secretory pathway at the late Golgi stage as reported by Novic, P., et al., Cell 21:205 (1980), the disclosure of which is incorporated herein by reference. Analysis of suppressors that permit the sec14.sup.ts mutant to grow at the restrictive temperature led to the discovery that mutations in the cytidinediphosphatecholine (CDP-choline) pathway for phosphatidylcholine (PC) biosynthesis (see, FIG. 1) suppress the sec14 mutant phenotype, allowing wild type growth even in strains carrying a total deletion of the SEC14 gene as reported by Cleves, A., et al. (1991).
The CDP-choline pathway, first described by Kennedy, E. P., et al. J. Biol. Chem. 222:193 (1956), the disclosure of which is incorporated herein by reference, and also known as the Kennedy pathway, is one of two routes for synthesis of PC in eukaryotic cells, including yeast. The second route for synthesis of PC, originally reported by Bremer, J., et al., Biochim. Biophys. Acta. 37:173 (1960), the disclosure of which is incorporated herein by reference, involves methylation of phosphatidylethanolamine (PE). Yeast cells can utilize either the CDP-choline or the PE methylation pathway, or a combination of the two, for net PC synthesis as reported by Griac, P., et al., J. Biol. Chem. 271:25692 (1996), the disclosure of which is incorporated herein by reference. (See, FIG. 1). Deletion of the genes in the CDP-choline pathway is not lethal in yeast and appears to have little effect on growth as reported by Griac, P., et al., and McMaster C. P. , et al. J. Biol. Chem. 269:14776 (1994), the disclosure of which is incorporated herein by reference. It has been widely assumed that the CDP-choline pathway in yeast functions largely for the utilization of exogenous choline. However, recent studies have suggested that the CDP-choline pathway contributes substantially to PC biosynthesis even in the absence of exogenous choline. See, McMaster, C. R., et al., J. Biol. Chem. 269:14776 (1994); McDonough, V. M., et al., J. Biol. Chem. 270(32):18774 (1995); and McGee, T. P., et al., J. Cell. Biol. 124:273 (1994), the disclosures of which are incorporated herein by reference. In the absence of exogenous choline, the yeast cell synthesizes PC predominantly via methylation of PE. Conversely, yeast cells can survive the complete and simultaneous deletion of the genes encoding the two phospholipid methyltransferases that carry out the three-step conversion of PE to PC, provided choline is supplied in the growth medium as reported by Summers, E. F., et al., Genetics 120:909 (1988), the disclosure of which is incorporated herein by reference.
However, deletions of the genes encoding enzymes in either of these two pathways result in subtly different phenotypes. For example, the deletion of either of the phospholipid methyltransferases does not suppress the sec14 growth phenotype as reported by Cleves, A., et al., Cell 64:789 (1991a) and Cleves, A., et al., Trends Cell. Bio. 1:30 (1991b), the disclosures of which are incorporated herein by reference. However, such mutants are unable to repress the INO1 gene that encodes inositol-1-phosphate synthase (see, FIG. 1) in response to exogenous inositol, unless PC biosynthesis is restored via the CDP-choline pathway. See, Summers, E. F., et al. (1988), and McGraw, P., et al., Genetics 122:317 (1989), the disclosure of which is incorporated herein by reference. And while the deletion of genes encodes enzymes of the CDP-choline pathway does not affect INO1 regulation in response to inositol (ee, Griac, P., et al. (1996)), such mutants suppress the sec14 phenotype (see, Cleves, A., et al. (1991a) and (1991b)).
The INO1 gene is the most highly regulated of a set of genes that encode enzymes of phospholipid biosynthesis that are subject to complex coordinate control. All these genes contain a conserved promoter element, UAS.sub.INO, that includes within it the canonical binding site, CANNTG, for transcription factors of the basic helix-loop-helix class as reported by Paltauf, F., et al., The Molecular and Cellular Biology of Yeast Saccharomvces (Broach, J., et al. eds.) Vol. II, pp. 415-500, Cold Spring Harbor Laboratory Press, Plainview, N.Y. (1992), and Bachhawat, N., et al., J. Biol. Chem. 270:25087 (1995), the disclosures of which are incorporated herein by reference.
There remains a need, however, for methods to control PC turnover and INO1 regulation in vivo in yeast in order to produce inositol and choline and other inositol- and choline-containing metabolites as well as phosphatidylinositol and phosphatidylcholine. There also remains a need for an improved test for detecting phospholipase D activity and the resulting choline in yeast cultures and a variety of other cell cultures.
According to the present invention, the identification of the metabolic signal (phosphatidic acid (PA)) for derepression of phospholipid biosynthesis in combination with the use of yeast strains which contain mutations that block PC biosynthesis and which are also mutated in their PI/PC transfer protein, SEC14, result in yeast strains that can produce enhanced amounts of inositol and choline as well as other phospholipid metabolites economically. The present invention also provides a means of detecting phospholipase D-mediated or other turnover of PC in vivo via a plate assay for choline excretion.